TGF-B and activin are functionally related members of the TGF-Beta superfamily of growth and differentiation factors. TGF-Beta and activin activate Smad2 and Smad3 and they both regulate tissue homeostasis by potently inhibiting proliferation of multiple cell types, including epithelial cells. Disruption of the antiproliferative response to activin and/or TGF-Beta is associated with dysregulated cellular proliferation, oncogenesis and progression toward the malignant phenotype. Cripto is a developmental oncoprotein that is overexpressed in multiple human tumors including approximately 80% of human breast tumors. Cripto overexpression transforms mammary epithelial ceils and activates the growth promoting Erk/MAPK and PI3K signaling pathways. Cripto and related EGF-CFC proteins also play essential signaling roles during embryonic development and Cripto is required for mesoderm induction and cardiogenesis. It has been shown that Cripto acts as a coreceptor to facilitate receptor assembly and signaling via activin type II/I receptors by the TGF-Beta superfamily members nodal, Vgl and GDF1. This led us to test whether Cripto may regulate the ability of activin and possibly other TGF-Beta ligands to assemble functional receptor complexes and we showed that Cripto binds activin in the presence of type II activin receptors, competes with ALK4 for binding to activin and antagonizes activin signaling. We now have evidence showing that Cripto also binds TGF-Beta in the presence of TBRII, competes with ALK5 for TGF-Beta binding and antagonizes TGF-Beta signaling; we propose, therefore, that Cripto may be generally capable of blocking antiproliferative Smad2/3 signals. Under Aim I of this proposal we will identify the molecular determinants on Cripto that mediate Cripto binding to activin/TFG-Beta and antagonism of activin/TGF-Beta signaling. We also propose to develop reagents including anti-Cripto neutralizing antibodies designed to prevent the ability of Cripto to antagonize activin and TGF-Beta signaling. Aim II proposes to characterize the ability of Cripto to block type I receptor recruitment to ligand-type II receptor complexes and block type I receptor phosphorylation in breast cancer cells in vitro in response to activin/TGF-Beta and the ability of Cripto to block phosphorylation of Smad2/3 following activin/TGF-beta treatment of breast cancer cells in vitro. Under Aim III we will characterize the effects of Cripto on activin/TGF-Beta-induced growth inhibition in three mammary epithelial-derived cell lines: MCF-7; MCF-10A; and CID-9 cells. Together, these studies will lead to an improved understanding of the mechanisms of Cripto action and will help illuminate therapeutic targets for the management of neoplastic disease.